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We present a suite of nine scenarios of future emissions trajectories of anthropogenic sources, a key deliverable of the ScenarioMIP experiment within CMIP6. Integrated assessment model results for 14 different emissions species and 13 emissions sectors are provided for each scenario with consistent transitions from the historical data used in CMIP6 to future trajectories using automated harmonization before being downscaled to provide higher emissions source spatial detail. We find that the scenarios span a wide range of end-of-century radiative forcing values, thus making this set of scenarios ideal for exploring a variety of warming pathways. The set of scenarios is bounded on the low end by a 1.9 W m−2 scenario, ideal for analyzing a world with end-of-century temperatures well below 2 ∘C, and on the high end by a 8.5 W m−2 scenario, resulting in an increase in warming of nearly 5 ∘C over pre-industrial levels. Between these two extremes, scenarios are provided such that differences between forcing outcomes provide statistically significant regional temperature outcomes to maximize their usefulness for downstream experiments within CMIP6. A wide range of scenario data products are provided for the CMIP6 scientific community including global, regional, and gridded emissions datasets.

Abstract Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD.

ABSTRACT Objectives Fusobacterium can contribute to oral diseases, but also pose as a systemic risk factor. This genus, and especially F. nucleatum, can be found in colorectal cancer (CRC) tissue and is involved in multiple aspects of this type of cancer. Previous studies indicated a possible oral origin of these bacteria; however, stronger evidence is needed to reach a definitive conclusion. This pilot study aimed to establish a method to successfully compare, at the strain level, fusobacteria from the oral cavity and CRC resection material for future cohort studies of CRC patients. Material and Methods In a first cohort of eight periodontitis patients, gingival crevicular fluid and saliva were collected. Fusobacterium was isolated on two different media. In a second cohort, saliva and CRC resection material were collected from ten CRC patients. These samples were used for screening of Fusobacterium with culturing, 16S rRNA gene profiling and a PCR‐based approach. Results In the first cohort, different Fusobacterium species were identified in GCF and saliva samples. However, as the total yield of Fusobacterium seemed slightly higher in saliva samples, it was therefore preferred for subsequent sample collection. Thus, in the second cohort, patient‐matched saliva and CRC resection material were screened for Fusobacterium and this showed that nine patients were culture‐positive in the saliva samples; however, no Fusobacterium could be isolated from the resection material. On the other hand, 16S rRNA gene profiling of the resection material indicated that eight CRC patients were positive for Fusobacterium. All eight of these patients carried Fusobacterium in their saliva, indicated by both marker gene PCR and culture‐based screening. Conclusions These pilot results are compatible with data from previous studies, indicating a possible link between oral and CRC‐associated Fusobacterium, and a more in‐depth analysis of specific strains and their characteristics in a larger cohort is justified. Trial Registration The protocol was registered at clinicaltrials.gov (NCT05945082).

IntroductionMaternal obesity poses risks for both mother and offspring during pregnancy, with underlying mechanisms remaining largely unexplored. Obesity is associated with microbial gut dysbiosis and low-grade inflammation, and also the diet has a major impact on these parameters. This study aimed to investigate how maternal obesity and diet contribute to changes in immune responses, exploring potential associations with gut microbiota dysbiosis and adverse pregnancy outcomes in mice.MethodsBefore mating, C57BL/6 mice were assigned to either a high-fat-diet (HFD) or low-fat-diet (LFD) to obtain obese (n=17) and lean (n=10) mice. To distinguish between the effects of obesity and diet, 7 obese mice were switched from the HFD to the LFD from day 7 until day 18 of pregnancy (“switch group”), which was the endpoint of the study. T helper (Th) cell subsets were studied in the spleen, mesenteric lymph nodes (MLN) and Peyer’s patches (PP), while monocyte subsets and activation status were determined in maternal blood (flow cytometry). Feces were collected before and during pregnancy (day 7,14,18) for microbiota analysis (16S rRNA sequencing). Pregnancy outcome included determination of fetal and placental weight.ResultsObesity increased splenic Th1 and regulatory T cells, MLN Th1 and PP Th17 cells and enhanced IFN-γ and IL-17A production by splenic Th cells upon ex vivo stimulation. Switching diet decreased splenic and PP Th2 cells and classical monocytes, increased intermediate monocytes and activation of intermediate/nonclassical monocytes. Obesity and diet independently induced changes in the gut microbiota. Various bacterial genera were increased or decreased by obesity or the diet switch. These changes correlated with the immunological changes. Fetal weight was lower in the obese than the lean group, while placental weight was lower in the switch than the obese group. DiscussionThis study demonstrates that obesity and diet independently impact peripheral and intestinal immune responses at the end of pregnancy. Simultaneously, both factors affect specific bacterial gut genera and lead to reduced fetal or placental weight. Our data suggest that switching diet during pregnancy to improve maternal health is not advisable and it supports pre/probiotic treatment of maternal obesity-induced gut dysbiosis to improve maternal immune responses and pregnancy outcome.

Background and aims: – Pseudoxanthoma elasticum (PXE) is a rare genetic disease caused by pathogenic mutations in the ABCC6 gene, resulting in low values of inorganic pyrophosphate (PPi). While low PPi is thought to contribute to arterial calcification, it remains unclear whether this fully explains premature calcification in PXE. It has been hypothesized that the ABCC6 gene could be related to dyslipidemia, which could contribute to vascular calcification seen in PXE. The aim of this study is to evaluate the relation between PXE and plasma lipid concentrations in a large cohort of PXE patients compared with reference values for the general population and compared with non-PXE controls. Methods: – The plasma concentrations of total cholesterol, HDL-cholesterol, tiglycerides, and LDL-cholesterol of 312 PXE patients were compared to age- and sex-matched modeled data of the general Dutch population. Differences in median lipid levels were compared with Mann-Whitney-U test. Secondly, plasma lipid concentrations of 44 PXE patients were compared to 44 not-genetically related relatives (spouses or friends), with linear models adjusted for age, sex and BMI. Results: – Total cholesterol in PXE patients was 5.6 [IQR 4.6–6.4] mmol/L versus 5.3 [IQR 4.7–6.0] mmol/L (p

Abstract The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome–phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies.

Abstract Disrupted host-microbe interactions at the mucosal level are key to the pathophysiology of IBD. This study aimed to comprehensively examine crosstalk between mucosal gene expression and microbiota in patients with IBD. To study tissue-specific interactions, we perform transcriptomic (RNA-seq) and microbial (16S-rRNA-seq) profiling of 697 intestinal biopsies (645 derived from 335 patients with IBD and 52 from 16 non-IBD controls). Mucosal gene expression patterns in IBD are mainly determined by tissue location and inflammation, whereas the mucosal microbiota composition shows a high degree of individual specificity. Analysis of transcript-bacteria interactions identifies six distinct groups of inflammation-related pathways that are associated with intestinal microbiota (adjusted P

ABSTRACT Gut dysbiosis has been associated with impaired outcomes in liver and kidney transplant recipients, but the gut microbiome of lung transplant recipients has not been extensively explored. We assessed the gut microbiome in 64 fecal samples from end-stage lung disease patients before transplantation and 219 samples from lung transplant recipients after transplantation using metagenomic sequencing. To identify dysbiotic microbial signatures, we analyzed 243 fecal samples from age-, sex-, and BMI-matched healthy controls. By unsupervised clustering, we identified five groups of lung transplant recipients using different combinations of immunosuppressants and antibiotics and analyzed them in relation to the gut microbiome. Finally, we investigated the gut microbiome of lung transplant recipients in different chronic lung allograft dysfunction (CLAD) stages and longitudinal gut microbiome changes after transplantation. We found 108 species (58.1%) in end-stage lung disease patients and 139 species (74.7%) in lung transplant recipients that were differentially abundant compared with healthy controls, with several species exhibiting sharp longitudinal increases from before to after transplantation. Different combinations of immunosuppressants and antibiotics were associated with specific gut microbial signatures. We found that the gut microbiome of lung transplant recipients in CLAD stage 0 was more similar to healthy controls compared to those in CLAD stage 1. Finally, the gut microbial diversity of lung transplant recipients remained lower than the average gut microbial diversity of healthy controls up to more than 20 years post-transplantation. Gut dysbiosis, already present before lung transplantation was exacerbated following lung transplantation.IMPORTANCEThis study provides extensive insights into the gut microbiome of end-stage lung disease patients and lung transplant recipients, which warrants further investigation before the gut microbiome can be used for microbiome-targeted interventions that could improve the outcome of lung transplantation.

Abstract Kidney transplant recipients (KTR) have impaired health-related quality of life (HRQoL) and suffer from intestinal dysbiosis. Increasing evidence shows that gut health and HRQoL are tightly related in the general population. Here, we investigate the association between the gut microbiome and HRQoL in KTR, using metagenomic sequencing data from fecal samples collected from 507 KTR. Multiple bacterial species are associated with lower HRQoL, many of which have previously been associated with adverse health conditions. Gut microbiome distance to the general population is highest among KTR with an impaired physical HRQoL (R = −0.20, P = 2.3 × 10−65) and mental HRQoL (R = −0.14, P = 1.3 × 10−3). Physical and mental HRQoL explain a significant part of variance in the gut microbiome (R 2 = 0.58%, FDR = 5.43 × 10−4 and R 2 = 0.37%, FDR = 1.38 × 10−3, respectively). Additionally, multiple metabolic and neuroactive pathways (gut brain modules) are associated with lower HRQoL. While the observational design of our study does not allow us to analyze causality, we provide a comprehensive overview of the associations between the gut microbiome and HRQoL while controlling for confounders.

Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nM affinity dependent on the species. G13 affinity was difficult to determine by biolayer interferometry due to low and heterogeneous signals. G13 and G6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species.

Abstract Cytotoxicity (i.e. cell death) is the core mechanism by which chemotherapy induces its anti-cancer effects. Unfortunately, this same mechanism underpins the collateral damage it causes to healthy tissues. The gastrointestinal tract is highly susceptible to chemotherapy’s cytotoxicity, resulting in ulcerative lesions (termed gastrointestinal mucositis, GI-M) that impair the functional capacity of the gut leading to diarrhea, anorexia, malnutrition and weight loss, which negatively impact physical/psychological wellbeing and treatment adherence. Preventing these side effects has proven challenging given the overlapping mechanisms that dictate chemotherapy efficacy and toxicity. Here, we report on a novel dietary intervention that, due to its localized gastrointestinal effects, is able to protect the intestinal mucosal from unwanted toxicity without impairing the anti-tumor effects of chemotherapy. The test diet (containing extensively hydrolyzed whey protein and medium chain triglycerides (MCTs)), was investigated in both tumor-naïve and tumor-bearing models to evaluate its effect on GI-M and chemo-efficacy, respectively. In both models, methotrexate was used as the representative chemotherapeutic agent and the diet was provided ad libitum for 14 days prior to treatment. GI-M was measured using the validated biomarker plasma citrulline, and chemo-efficacy defined by tumor burden (cm3/g body weight). The test diet significantly attenuated GI-M (P = 0.03), with associated reductions in diarrhea (P

IntroductionIntestinal epithelial cells produce interleukin-18 (IL-18), a key factor in promoting epithelial barrier integrity. Here, we analyzed the potential role of gut bacteria and the hypoxia-inducible factor 1α (HIF1α) pathway in regulating mucosal IL18 expression in inflammatory bowel disease (IBD).MethodsMucosal samples from patients with IBD (n = 760) were analyzed for bacterial composition, IL18 levels and HIF1α pathway activation. Wild-type Caco-2 and CRISPR/Cas9-engineered Caco-2-HIF1A-null cells were cocultured with Faecalibacterium prausnitzii in a “Human oxygen-Bacteria anaerobic” in vitro system and analyzed by RNA sequencing.ResultsMucosal IL18 mRNA levels correlated positively with the abundance of mucosal-associated butyrate-producing bacteria, in particular F. prausnitzii, and with HIF1α pathway activation in patients with IBD. HIF1α-mediated expression of IL18, either by a pharmacological agonist (dimethyloxallyl glycine) or F. prausnitzii, was abrogated in Caco-2-HIF1A-null cells.ConclusionButyrate-producing gut bacteria like F. prausnitzii regulate mucosal IL18 expression in a HIF1α-dependent manner that may aid in mucosal healing in IBD.

The objective was to assess the effect of peri-implantitis surgery on the peri-implant microbiome with a follow-up of one year. A total of 25 peri-implantitis patients in whom non-surgical treatment has failed to solve peri-implantitis underwent resective surgical treatment. Their peri-implant pockets were sampled prior to surgical treatment (T0) and one year post treatment (T12). The natural dentition was sampled to analyse similarities and differences with the peri-implantitis samples. Treatment success was recorded. The change in microbial relative abundance levels was evaluated. The microbiota was analysed by sequencing the amplified V3-V4 region of the 16S rRNA genes. Sequence data were binned to amplicon sequence variants that were assigned to bacterial genera. Group differences were analysed using principal coordinate analysis, Wilcoxon signed rank tests, and t-tests. Beta diversity analyses reported a significant separation between peri-implantitis and natural dentition samples on T0 and T12, along with significant separations between successfully and non-successfully treated patients. Eubacterium was significantly lower on T12 compared to T0 for the peri-implantitis samples. Treponema and Eubacterium abundance levels were significantly lower in patients with treatment success on T0 and T12 versus no treatment success. Therefore, lower baseline levels of Treponema and Eubacterium seem to be associated with treatment success of peri-implantitis surgery. This study might aid clinicians in determining which peri-implantitis cases might be suitable for treatment and give a prognosis with regard to treatment success.

Background: Scapular notching continues to be associated with reverse shoulder arthroplasty (RSA) and is thought to lead to fewer outcomes. Decreasing the humeral neck-shaft angle (NSA) has been associated with decreased incidence of scapular notching. Lateralizing the glenosphere center of rotation (COR) has also been proposed to decrease notching; however, its effect in lower NSA RSA is less understood. The purpose of this study was to compare the impact of the medial (0 mm) and lateral (4 mm) COR on the incidence of scapular notching and clinical outcomes after RSA with a 135° NSA humeral component. Methods: We performed a multicenter retrospective comparative cohort of 82 patients with cuff tear arthropathy (41 in each cohort) who underwent RSA with a 135° NSA humeral component and a glenosphere COR of either 0 mm (medialized COR [MCOR]) or 4 mm (lateralized COR [LCOR]) of lateralization. RSA was performed using the same 135° humeral system and baseplate design. All patients had 2-year radiographic and clinical follow-up. Postoperative radiographs were evaluated for scapular notching. Clinical outcomes included American Shoulder and Elbow Surgeons scores, visual analog pain scale, Simple Assessment Numeric Evaluation, and active range of motion. Results: The overall incidence of scapular notching was 22.0%. There was no significant difference in scapular notching between cohorts: 24.4% in the MCOR and 19.5% in the LCOR (P = .625). Both cohorts had significant improvements in American Shoulder and Elbow Surgeons scores, visual analog pain scale, Simple Assessment Numeric Evaluation, and active range of motion postoperatively (P

Background Diet plays a pivotal role in the onset and progression of Crohn’s disease (CD). Nutritional interventions revealed effects on intestinal inflammation and gut microbial composition. However, data from well-designed and controlled dietary trials are lacking. Therefore, evidence-based dietary recommendations are still unavailable to patients and physicians. Here, we aim to investigate the effects of an evidence-based anti-inflammatory diet, and an ileocolonic-targeted capsule containing vitamin B2, B3 and C (ColoVit) on patients with CD and their healthy household members.Methods and analysis In this multicentre, randomised, placebo-controlled, partially blinded nutritional intervention trial, we aim to recruit 255 CD patients with Harvey-Bradshaw Index

BackgroundIn the previously reported SAPS trial (https://clinicaltrials.gov/ct2/show/NCT01139489), procalcitonin-guidance safely reduced the duration of antibiotic treatment in critically ill patients. We assessed the impact of shorter antibiotic treatment on antimicrobial resistance development in SAPS patients.Materials and methodsCultures were assessed for the presence of multi-drug resistant (MDR) or highly resistant organisms (HRMO) and compared between PCT-guided and control patients. Baseline isolates from 30 days before to 5 days after randomization were compared with those from 5 to 30 days post-randomization. The primary endpoint was the incidence of new MDR/HRMO positive patients.ResultsIn total, 8,113 cultures with 96,515 antibiotic test results were evaluated for 439 and 482 patients randomized to the PCT and control groups, respectively. Disease severity at admission was similar for both groups. Median (IQR) durations of the first course of antibiotics were 6 days (4–10) and 7 days (5–11), respectively (p = 0.0001). Antibiotic-free days were 7 days (IQR 0–14) and 6 days (0–13; p = 0.05). Of all isolates assessed, 13% were MDR/HRMO positive and at baseline 186 (20%) patients were MDR/HMRO-positive. The incidence of new MDR/HRMO was 39 (8.9%) and 45 (9.3%) in PCT and control patients, respectively (p = 0.82). The time courses for MDR/HRMO development were also similar for both groups (p = 0.33).ConclusionsIn the 921 randomized patients studied, the small but statistically significant reduction in antibiotic treatment in the PCT-group did not translate into a detectable change in antimicrobial resistance. Studies with larger differences in antibiotic treatment duration, larger study populations or populations with higher MDR/HRMO incidences might detect such differences.

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.

Abstract Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005–50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

Abstract Background Vancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome’s structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates. Results Analysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity. Conclusions For rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period.

Fecal microbiota transplantation (FMT) is under investigation for several indications, including ulcerative colitis (UC). The clinical success of FMT depends partly on the engraftment of viable bacteria. Because the vast majority of human gut microbiota consists of anaerobes, the currently used aerobic processing protocols of donor stool may diminish the bacterial viability of transplanted material. This study assessed the effect of four processing techniques for donor stool (i.e., anaerobic and aerobic, both direct processing and after temporary cool storage) on bacterial viability. By combining anaerobic culturing on customized media for anaerobes with 16S rRNA sequencing, we could successfully culture and identify the majority of the bacteria present in raw fecal suspensions. We show that direct anaerobic processing of donor stool is superior to aerobic processing conditions for preserving the bacterial viability of obligate anaerobes and butyrate-producing bacteria related to the clinical response to FMT in ulcerative colitis patients, including Faecalibacterium, Eubacterium hallii, and Blautia. The effect of oxygen exposure during stool processing decreased when the samples were stored long-term. Our results confirm the importance of sample conditioning to preserve the bacterial viability of oxygen-sensitive gut bacteria. Anaerobic processing of donor stool may lead to increased clinical success of FMT, which should further be investigated in clinical trials.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with KD of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (

Abstract Background The intestinal microbiome in preterm infants differs markedly from term infants. It is unclear whether the microbiome develops over time according to infant specific factors. Methods We analysed (clinical) metadata - to identify the main factors influencing the microbiome composition development - and the first meconium and faecal samples til the 4th week via 16 S rRNA amplican sequencing. Results We included 41 infants (gestational age 25–30 weeks; birth weight 430-990 g. Birth via Caesarean section (CS) was associated with placental insufficiency during pregnancy and lower BW. In meconium samples and in samples from weeks 2 and 3 the abundance of Escherichia and Bacteroides (maternal faecal representatives) were associated with vaginal delivery while Staphylococcus (skin microbiome representative) was associated with CS. Secondly, irrespective of the week of sampling or the mode of birth, a transition was observed as children children gradually increased in weight from a microbiome dominated by Staphylococcus (Bacilli) towards a microbiome dominated by Enterobacteriaceae (Gammaproteobacteria). Conclusions Our data show that the mode of delivery affects the meconium microbiome composition. They also suggest that the weight of the infant at the time of sampling is a better predictor for the stage of progression of the intestinal microbiome development/maturation than postconceptional age as it less confounded by various infant-specific factors.

IntroductionInflammatory bowel disease (IBD) is characterized by a disturbed gut microbiota composition. Patients with IBD have both elevated mucosal and serum levels of IgG-antibodies directed against bacterial antigens, including flagellins. In this study, we aimed to determine to which intestinal bacteria the humoral immune response is directed to in patients with IBD.MethodsFecal and serum samples were collected from patients with IBD (n=55) and age- and sex-matched healthy controls (n=55). Fecal samples were incubated with autologous serum and IgG-coated fractions were isolated by magnetic-activated cell sorting (MACS) and its efficiency was assessed by flow cytometry. The bacterial composition of both untreated and IgG-coated fecal samples was determined by 16S rRNA-gene Illumina sequencing.ResultsIgG-coated fecal samples were characterized by significantly lower microbial diversity compared to the fecal microbiome. Both in patients with IBD and controls, serum IgG responses were primarily directed to Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Veillonella and Enterobacteriaceae, as well as against specific Lachnospiraceae bacteria, including Coprococcus and Dorea (all P

Alzheimer's disease (AD) is a global public health priority as with aging populations, its prevalence is expected to rise even further in the future. The brain and gut are in close communication through immunological, nervous and hormonal routes, and therefore, probiotics are examined as an option to influence AD hallmarks, such as plaques, tangles, and low grade inflammation. This study aimed to provide an overview of the available animal evidence on the effect of different probiotics on gut microbiota composition, short chain fatty acids (SCFAs), inflammatory markers, Amyloid-β (Aβ), and cognitive functioning in AD animal models. A systematic literature search was performed in PubMed, SCOPUS, and APA PsychInfo. Articles were included up to May 2021. Inclusion criteria included a controlled animal study on probiotic supplementation and at least one of the abovementioned outcome variables. Of the eighteen studies, most were conducted in AD male mice models (n = 9). Probiotics of the genera Lactobacillus and Bifidobacterium were used most frequently. Probiotic administration increased species richness and/or bacterial richness in the gut microbiota, increased SCFAs levels, reduced inflammatory markers, and improved cognitive functioning in AD models in multiple studies. The effect of probiotic administration on Aβ remains ambiguous. B. longum (NK46), C. butyricum, and the mixture SLAB51 are the most promising probiotics, as positive improvements were found on almost all outcomes. The results of this animal review underline the potential of probiotic therapy as a treatment option in AD.

Background: In shoulder arthroplasty, cerclage fixation techniques are used to stabilize osteotomies, fractures, and allografts. Fixation techniques including cerclage with metal and polymer cables have been described. The purpose of this study was to evaluate suture cerclage fixation of the humeral shaft during shoulder arthroplasty. Materials and methods: Shoulder arthroplasty cases performed from 2012-2017 by 3 fellowship-trained shoulder arthroplasty surgeons were reviewed. Cases in which suture cerclage was used for osteotomy, fracture, or allograft fixation were identified. Demographic data, indications, implant types, fixation techniques, and complications were reviewed. Postoperative radiographs were analyzed for union, component loosening, and loss of fixation. American Shoulder and Elbow Surgeons and Single Assessment Numeric Evaluation scores were collected preoperatively and postoperatively. Results: A total of 27 patients (3 primary and 24 revision cases) with a mean age of 69.6 years (range, 28-88 years) were available for follow-up at a mean of 12.6 months (range, 0.8-42.3 months) postoperatively. Humeral osteotomy alone was performed in 15 cases. Allograft alone was used in 1 case. Both allograft and osteotomy were used in 6 cases. Complications occurred in 3 patients (11%), comprising 1 postoperative periprosthetic fracture and 2 prosthetic joint infections; all required further surgery. Radiographs at final follow-up showed healing of all osteotomies and fractures. No hardware failure or implant loosening occurred. The mean postoperative American Shoulder and Elbow Surgeons and Single Assessment Numeric Evaluation scores were significantly improved (α = .05) compared with preoperative scores, from 21.4 to 44.5 (P = .002) and from 26.7 to 74.1 (P < .001), respectively. Conclusion: Suture cerclage is safe and effective for humeral fixation in shoulder arthroplasty.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis.Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content.Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster’s closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes.Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients.

Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD

Abstract Gut microbiota development in formula‐fed and breast‐fed infants is known to differ. This could relate to the usage of unmodified vegetable oil instead of mammalian fat in infant formula (IF), causing the enhanced formation of the poorly soluble soap calcium palmitate (CP) in the infant's gut. Here we investigate in vitro the possible influence of CP on the infant gut bacteria. The growth of several bacterial species dominant in the infant's gut was analyzed by culturing in media with CP. Faecalibacterium prausnitzii as a sensitive representative was analyzed in detail by scanning transmission electron microscopy, membrane staining, gas chromatography, and microbial fuel cell experiments. Of all bacteria tested, the growth of several bifidobacteria and F. prausnitzii was reduced at 0.01 mg/ml CP, Bifidobacterium infantis stopped growing completely. CP reduced the cell envelope thickness of F. prausnitzii, disturbed the cell membrane fatty acids and function of membrane proteins involved in electron transport. CP inhibited the growth of bifidobacteria and faecalibacteria. This suggests that modification of fat in IF may benefit the development of the gut microbiota in formula‐fed infants by supporting the colonization of important beneficial bacteria in early life. Future clinical studies are needed to confirm this.

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127−/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.

Many chronic diseases are associated with decreased abundance of the gut commensal Faecalibacterium prausnitzii. This strict anaerobe can grow on dietary fibers, e.g., prebiotics, and produce high levels of butyrate, often associated to epithelial metabolism and health. However, little is known about other F. prausnitzii metabolites that may affect the colonic epithelium. Here, we analyzed prebiotic cross-feeding between F. prausnitzii and intestinal epithelial (Caco-2) cells in a “Human-oxygen Bacteria-anaerobic” coculture system. Inulin-grown F. prausnitzii enhanced Caco-2 viability and suppressed inflammation- and oxidative stress-marker expression. Inulin-grown F. prausnitzii produced excess butyrate and fructose, but only fructose efficiently promoted Caco-2 growth. Finally, fecal microbial taxonomy analysis (16S sequencing) from healthy volunteers (n = 255) showed the strongest positive correlation for F. prausnitzii abundance and stool fructose levels. We show that fructose, produced and accumulated in a fiber-rich colonic environment, supports colonic epithelium growth, while butyrate does not.

Background Recent findings by Tang et al. (2020) show dietary restriction (30%, 2 weeks) prevents methotrexate-induced mortality by modulation of the microbiota, specifically the expansion of Lactobacillus. While fundamentally insightful, upscaling this schedule is a major obstacle to clinical uptake. Here, we evaluate a safe and clinically achievable schedule of pre-therapy fasting for 48 h on microbiota composition, body composition and intestinal proliferation, and assess its impact on the severity of methotrexate-induced gastrointestinal mucositis using a validated preclinical rat model. Methods Age- and weight-matched male Wistar rats were treated with a sublethal dose of 45 mg/kg methotrexate with or without pre-therapy fasting. The impact of acute fasting on epithelial proliferation, body composition and the microbiota was assessed using plasma citrulline, Ki67 immunohistochemistry, miniSpec and 16S rRNA sequencing. The severity of gastrointestinal mucositis was evaluated using plasma citrulline and body weight. Results Whilst pre-therapy fasting slowed epithelial proliferation and increased microbial diversity and richness, it also induced significant weight loss and was unable to attenuate the severity of mucositis in both age- and weight-matched groups. In contrast to Tang et al., we saw no expansion of Lactobacillus following acute fasting. Conclusions Our findings suggest that the beneficial effects of acute fasting are masked by the detrimental effects on body weight and composition and lacking influence on Lactobacillus. Future studies should consider alternative fasting schedules or aim to induce comparable microbial and mucosal manipulation without compromising body composition using clinically feasible methods of dietary or microbial intervention.

Antimicrobial prophylaxis is increasingly being used in patients with hematological malignancies receiving high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT). However, few studies have focused on the potential impact of gastrointestinal mucositis (GI-M), a frequently observed side effect of chemotherapy in patients with cancer that affects the gastrointestinal microenvironment, on drug absorption. In this review, we discuss how chemotherapy leads to an overall loss of mucosal surface area and consequently to uncontrolled transport across the barrier. The barrier function is depending on intestinal luminal pH, intestinal motility, and diet. Another factor contributing to drug absorption is the gut microbiota, as it modulates the bioavailability of orally administrated drugs by altering the gastrointestinal properties. To better understand the complex interplay of factors in GI-M and drug absorption we suggest: (i) the longitudinal characterization of the impact of GI-M severity on drug exposure in patients, (ii) the development of tools to predict drug absorption, and (iii) strategies that allow the support of the gut microbiota. These studies will provide relevant data to better design strategies to reduce the severity and impact of GI-M in patients with cancer.

Gut microbes are crucial to human health, but microbial composition is often disturbed in a number of human diseases. Accumulating evidence points to nutritional modulation of the gut microbiota as a potentially beneficial therapeutic strategy. Vitamin C (ascorbic acid) may be of particular interest as it has known antioxidant and anti-inflammatory properties. In this study, we investigated whether supplementation with high-dose vitamin C may favourably affect the composition of the gut microbiota. In this pilot study, healthy human participants received 1000 mg vitamin C supplementation daily for two weeks. Gut microbiota composition was analysed before and after intervention by performing faecal 16S rRNA gene sequencing. In total, 14 healthy participants were included. Daily supplementation of high-dose vitamin C led to an increase in the relative abundances of Lachnospiraceae (p < 0.05), whereas decreases were observed for Bacteroidetes (p < 0.01), Enterococci (p < 0.01) and Gemmiger formicilis (p < 0.05). In addition, trends for bacterial shifts were observed for Blautia (increase) and Streptococcus thermophilus (decrease). High-dose vitamin C supplementation for two weeks shows microbiota-modulating effects in healthy individuals, with several beneficial shifts of bacterial populations. This may be relevant as these bacteria have anti-inflammatory properties and strongly associate with gut health.

Abstract The establishment of the infant gut microbiota is a highly dynamic process dependent on extrinsic and intrinsic factors. We characterized the faecal microbiota of 4 breastfed infants and 4 formula-fed infants at 17 consecutive time points during the first 12 weeks of life. Microbiota composition was analysed by a combination of 16S rRNA gene sequencing and quantitative PCR (qPCR). In this dataset, individuality was a major driver of microbiota composition (P = 0.002) and was more pronounced in breastfed infants. A developmental signature could be distinguished, characterized by sequential colonisation of i) intrauterine/vaginal birth associated taxa, ii) skin derived taxa and other typical early colonisers such as Streptococcus and Enterobacteriaceae, iii) domination of Bifidobacteriaceae, and iv) the appearance of adultlike taxa, particularly species associated with Blautia, Eggerthella, and the potential pathobiont Clostridium difficile. Low abundance of potential pathogens was detected by 16S profiling and confirmed by qPCR. Incidence and dominance of skin and breast milk associated microbes were increased in the gut microbiome of breastfed infants compared to formula-fed infants. The approaches in this study indicate that microbiota development of breastfed and formula-fed infants proceeds according to similar developmental stages with microbiota signatures that include stage-specific species.

BACKGROUND:Intestinal permeability can be assessed by monitoring renal excretion of orally administered radioactively 51Cr-labeled ethylenediaminetetraacetic acid (51Cr-EDTA). Although considered safe, patient participation in using radio-labeled tracers is low. Here, we used orally administered 52Cr-EDTA as non-radioactive alternative to assess intestinal permeability in CD and analyzed the association with disease activity, disease location and gut microbial dysbiosis. MATERIALS AND METHODS:60 CD patients with low (n = 25) and increased (n = 35) fecal calprotectin levels (cut-off: 100 μg/g feces) ingested 20 mL 52Cr-EDTA (20 mmol/L) solution whereafter 24-h urine was collected. Urinary 52Cr-EDTA concentrations were quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Fecal Enterobacteriaceae and Faecalibacterium prausnitzii were quantified using FISH. Correlations between urinary 52Cr-EDTA excretion and other parameters were established using nonparametric Spearman's correlation coefficients (ρ). RESULTS:CD patients with increased fecal calprotectin levels (> 100 μg/g) demonstrated an elevated urinary 52Cr-EDTA/creatinine ratio (772 vs. 636 μmol/mol, P = 0.132). Patients with primarily colonic disease showed the highest 52Cr-EDTA excretion. Importantly, a positive correlation was observed for the urinary 52Cr-EDTA/creatinine ratio and fecal calprotectin levels (ρ = 0.325, P < 0.05). Finally, urinary 52Cr-EDTA/creatinine ratio negatively correlated with the relative abundance of Faecalibacterium prausnitzii (ρ = -0.221, P = 0.092), while positively correlating with Enterobacteriaceae (ρ = 0.202, P = 0.126). CONCLUSIONS:Orally administered and renal excreted 52Cr-EDTA may be used to assess intestinal permeability in CD and correlates with fecal calprotectin levels and bacterial species relevant to CD. This test may improve non-invasive detection of disease exacerbations and help monitor disease activity.

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

Integrated assessment models (IAMs) have emerged as key tools for building and assessing long term climate mitigation scenarios. Due to their central role in the recent IPCC assessments, and international climate policy analyses more generally, and the high uncertainties related to future projections, IAMs have been critically assessed by scholars from different fields receiving various critiques ranging from adequacy of their methods to how their results are used and communicated. Although IAMs are conceptually diverse and evolved in very different directions, they tend to be criticised under the umbrella of ‘IAMs’. Here we first briefly summarise the IAM landscape and how models differ from each other. We then proceed to discuss six prominent critiques emerging from the recent literature, reflect and respond to them in the light of IAM diversity and ongoing work and suggest ways forward. The six critiques relate to (a) representation of heterogeneous actors in the models, (b) modelling of technology diffusion and dynamics, (c) representation of capital markets, (d) energy-economy feedbacks, (e) policy scenarios, and (f) interpretation and use of model results.

Mucositis is a common side-effect of chemotherapy treatment, inducing alterations in the composition of the gut microbiota. Redox active compounds, such as vitamins B2 and C, have been shown to reduce inflammation and enhance the growth of anaerobic bacteria in the gut. We therefore aimed to (1) validate the ability of these compounds to promote bacterial cell growth in vitro, and (2) determine their prophylactic efficacy in a rat model of methotrexate (MTX)-induced mucositis. Bacterial growth curves were performed to assess the growth kinetics of bacteria exposed to Vitamins C and B2 (0.5 mM). Male wistar rats (150–200 g) received vitamins B2 (12 mg/day) and C (50 mg/day) via daily oral gavage (from day −1 to day 10). MTX (45 mg/Kg) was administrated via I.V. injection (N = 4–8/group) on day 0. Body weight, water/food consumption and diarrhea were assessed daily. Blood and faecal samples were collected longitudinally to assess citrulline levels (mucositis biomarker) and gut microbiota composition. Vitamins C/B2 enhanced the in vitro growth of anaerobic bacteria Blautia coccoides and Roseburia intestinalis. Contrarily to vitamin B2, in vivo administration of Vitamin C significantly attenuated clinical symptoms of mucositis. Despite their influence on the composition of the gut microbiota, both vitamins did not modulate the course of MTX-induced mucositis, as accessed by plasma citrulline. Vitamins B2 and C enhanced anaerobic bacterial growth in vitro, however their ability to mitigate MTX-induced mucositis was limited.